Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

Islas LD, Sigworth FJ - J. Gen. Physiol. (1999)

Bottom Line:
We find that Shab has a relatively small gating charge, approximately 7.5 e(o).Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1.Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACTThe members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

Figure 8: Substate kinetics in Shab channels. (A) Current traces at −70 mV from a patch containing three channels. The different horizontal dotted lines indicate the closed, substate, and full open levels. Note the various types of transitions indicated by the numbers. From the substate, a channel can either (1) close or (2) proceed to the fully open state; direct transitions from closed to fully open also occur (3). (B) All-point amplitude histograms from 200 sweeps at the indicated membrane potential. The histograms were fitted to a sum of three Gaussians and the individual components are shown. The letters identify the closed, substate, and fully open states according to the current amplitude. Absolute probabilities of being in the substate are: 0.078 at −70 mV, 0.012 at −80 mV, 0.0084 at −90 mV, and 0.021 at 10 mV. The probability of being in the fully open state are: 5.2 × 10−2 at −70 mV, 1.4 × 10−3 at −80 mV, 1.4 × 10−4 at −90 mV, and 0.75 at 10 mV. The histograms are from a patch containing three channels, except for the histogram at 10 mV, which is from a single-channel patch. (C) Steady state occupancies of the open (filled symbols) and subconductance (open symbols) states. Squares are data obtained from a one-channel patch in which the external K+ concentration [K+]o = 10 mM, and circles are from a multiple channel patch with [K+]o = 60 mM. Points from the low [K+]o patch have been shifted by −20 mV to approximately compensate for the shift in activation due to altered external potassium. The continuous curve represents the open-state probability as a function of voltage and the dotted curve is the probability of the substate, computed from the scheme shown, where the equilibrium constants at 0 mV and their effective charges are: K1 = 1.18, 1.2 eo; K2 = 1.18, 0.6 eo; K3 = 2.4, 1.8 eo. (D) Kinetic description of substate and opening transitions at negative potentials. Values of rate constants at −70 mV are given; in cases where a clear voltage dependence could be discerned over the voltage range of −90 to −65 mV, partial charges are also given in parentheses. The first latency to channel opening is roughly accounted for by the rate from state C to Cn (arrow at top left). Dotted arrows indicate transitions that occur but whose rates we could not determine.

Mentions:
Interestingly, single Shab channels in single-channel patches and in recordings from multiple channel patches show a tendency to open to subconductance states. These events arise from Shab channels since direct transitions are observed between the subconductance and the fully open states. Fig. 8 A shows representative current recordings at −70 mV. Indicated are the fully open state and the substate level at 40% of the full open current level. It is evident that the channel can dwell only in the substate or make transitions to the open state with or without having to traverse the substate level.

Figure 8: Substate kinetics in Shab channels. (A) Current traces at −70 mV from a patch containing three channels. The different horizontal dotted lines indicate the closed, substate, and full open levels. Note the various types of transitions indicated by the numbers. From the substate, a channel can either (1) close or (2) proceed to the fully open state; direct transitions from closed to fully open also occur (3). (B) All-point amplitude histograms from 200 sweeps at the indicated membrane potential. The histograms were fitted to a sum of three Gaussians and the individual components are shown. The letters identify the closed, substate, and fully open states according to the current amplitude. Absolute probabilities of being in the substate are: 0.078 at −70 mV, 0.012 at −80 mV, 0.0084 at −90 mV, and 0.021 at 10 mV. The probability of being in the fully open state are: 5.2 × 10−2 at −70 mV, 1.4 × 10−3 at −80 mV, 1.4 × 10−4 at −90 mV, and 0.75 at 10 mV. The histograms are from a patch containing three channels, except for the histogram at 10 mV, which is from a single-channel patch. (C) Steady state occupancies of the open (filled symbols) and subconductance (open symbols) states. Squares are data obtained from a one-channel patch in which the external K+ concentration [K+]o = 10 mM, and circles are from a multiple channel patch with [K+]o = 60 mM. Points from the low [K+]o patch have been shifted by −20 mV to approximately compensate for the shift in activation due to altered external potassium. The continuous curve represents the open-state probability as a function of voltage and the dotted curve is the probability of the substate, computed from the scheme shown, where the equilibrium constants at 0 mV and their effective charges are: K1 = 1.18, 1.2 eo; K2 = 1.18, 0.6 eo; K3 = 2.4, 1.8 eo. (D) Kinetic description of substate and opening transitions at negative potentials. Values of rate constants at −70 mV are given; in cases where a clear voltage dependence could be discerned over the voltage range of −90 to −65 mV, partial charges are also given in parentheses. The first latency to channel opening is roughly accounted for by the rate from state C to Cn (arrow at top left). Dotted arrows indicate transitions that occur but whose rates we could not determine.

Mentions:
Interestingly, single Shab channels in single-channel patches and in recordings from multiple channel patches show a tendency to open to subconductance states. These events arise from Shab channels since direct transitions are observed between the subconductance and the fully open states. Fig. 8 A shows representative current recordings at −70 mV. Indicated are the fully open state and the substate level at 40% of the full open current level. It is evident that the channel can dwell only in the substate or make transitions to the open state with or without having to traverse the substate level.

Bottom Line:
We find that Shab has a relatively small gating charge, approximately 7.5 e(o).Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1.Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

Affiliation:
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACTThe members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.